The interaction in vivo of transferrin and asialotransferrin with liver cells.

Rat transferrin or asialotransferrin doubly radiolabelled with 59Fe and 125I was injected into rats. A determination of extrahepatic and hepatic uptake indicated that asialotransferrin delivers a higher fraction of the injected 59Fe to the liver than does transferrin. In order to determine in vivo the intrahepatic recognition sites for transferrin and asialotransferrin, the liver was subfractionated into parenchymal, endothelial and Kupffer cells by a low-temperature cell isolation procedure. High-affinity recognition of transferrin (competed for by an excess of unlabelled transferrin) is exerted by parenchymal cells as well as endothelial and Kupffer cells with a 10-fold higher association (expressed per mg of cell protein) to the latter cell types. In all three cell types iron delivery occurs, as concluded from the increase in cellular 59Fe/125I ratio at prolonged circulation times of transferrin. It can be calculated that parenchymal cells are responsible for 50-60% of the interaction of transferrin with the liver, 20-30% is associated with endothelial cells and about 20% with Kupffer cells. For asialotransferrin a higher fraction of the injected dose becomes associated with parenchymal cells as well as with endothelial and Kupffer cells. Competition experiments in vivo with various sugars indicated that the increased interaction of asialotransferrin with parenchymal cells is specifically inhibited by N-acetylgalactosamine whereas mannan specifically inhibits the increased interaction of asialotransferrin with endothelial and Kupffer cells. Recognition of asialotransferrin by galactose receptors from parenchymal cells or mannose receptors from endothelial and Kupffer cells is coupled to active 59Fe delivery to the cells. It is concluded that, as well as parenchymal cells, liver endothelial and Kupffer cells are also quantitatively important intrahepatic sites for transferrin and asialotransferrin metabolism, an interaction exerted by multiple recognition sites on the various cell types.     

Influence of N6-methylation of residue A(5) on the conformational behaviour of d(C-C-G-A-A-T-T-C-G-G) in solution studied by 1H-NMR spectroscopy. 2. The hairpin form

The solution conformations of the oligonucleotides d(C-C-G-A-A-T-T-C-G-G) and d(C-C-G-A-m6A-T-T-C-G-G) as a function of temperature and sample concentration were investigated by means of 1H-NMR spectroscopy. The NMR spectra revealed that, at certain combinations of temperature and low sample and salt concentration, both compounds exist as a B-DNA-type duplex slowly (on the 1H-NMR time scale) interconverting with a monomeric species. From chemical shift data and imino-proton spectra, it is concluded that the monomeric species consists of a mixture of a hairpin form in rapid equilibrium with the random-coil form. The double-helical stem of the hairpin is formed by the six terminal cytidine and guanine residues, whereas the four core residues, -A-(m6)A-T-T-, partake in the loop. Thermodynamic analysis of the chemical shift of the resonances of the monomeric species vs temperature profiles of the two decamers and mutual comparison of these profiles indicate the following: the influence of N6-methylation of residue A(5) upon the local structure of the hairpin must be small; methylation decreases the stability of the duplex relative to the monomeric species: the temperature at which the fraction duplex equals 0.5 was found to be 312 K for the parent compound and 305 K for the methylated decamer at 2 mM sample concentration; methylation does not significantly alter the stability of the hairpin form relative to the random coil form: the Tm of the hairp----n equilibrium random-coil equilibrium is 308 K for the parent compound and 306 K for the methylated decamer. A higher fraction hairpin-like structure for the N6-methylated compound is observed under identical conditions of temperature and sample concentration: at 300 K, 2 mM sample concentration, the fraction hairpin form is 0.12 for d(C-C-G-A-A-T-T-C-G-G) and 0.20 for d(C-C-G-A-m6A-T-T-C-G-G). This finding appears to be a consequence of the reduced stability of the methylated dimeric species relative to the monomeric species, and to depend upon the sodium-ion concentration: it becomes more pronounced under low-salt conditions.     

Peroxisomes and peroxisomal functions in muscle. Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.     

Application of a reference convolution method to tryptophan fluorescence in proteins. A refined description of rotational dynamics

A reference method for the deconvolution of polarized fluorescence decay data is described. Fluorescence lifetime determinations for p-terphenyl, p-bis[2-(5-phenyloxazolyl)]benzene and N-acetyltryptophanamide (AcTrpNH2) show that with this method more reliable fits of the decays can be made than with the scatterer method, which is most frequently used. Analysis of the AcTrpNH2 decay with p-terphenyl as the reference compound yields an excellent fit with lifetimes of 2.985 ns for AcTrpNH2 and 1.099 ns for p-terphenyl (20 degrees C), whereas the AcTrpNH2 decay cannot be satisfactorily fitted when the scatterer method is used. The frequency of the detected photons is varied to determine the conditions where pulse pile-up starts to affect the measured decays. At detection frequencies of 5 kHz and 15 kHz, which corresponds to 1.7% and 5% respectively of the rate of the excitation photons no effects are found. Decays measured at 30 kHz (10%) are distorted, indicating that pile-up effects play a role at this frequency. The fluorescence and fluorescence anisotropy decays of the tryptophan residues in the proteins human serum albumin, horse liver alcohol dehydrogenase and lysozyme have been reanalysed with the reference method. The single tryptophan residue of the albumin is shown to be characterized by a triple-exponential fluorescence decay. The anisotropy decay of albumin was found to be mono-exponential with a rotational correlation time of 26 ns (20 degrees C). The alcohol dehydrogenase has two different tryptophan residues to which single lifetimes are assigned. It is found that the rotational correlation time for the dehydrogenase changes with excitation wavelength (33 ns for lambda ex = 295 nm and 36 ns for lambda ex = 300 nm at 20 degrees C), indicating a nonspherical protein molecule. Lysozyme has six tryptophan residues, which give rise to a triple-exponential fluorescence decay. A single-exponential decay with a rotational correlation time of 3.8 ns is found for the anisotropy. This correlation time is significantly shorter than that arising from the overall rotation and probably originates from intramolecular, segmental motion.     

Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.     

Heparan sulfate proteoglycans of human lung fibroblasts. Structural heterogeneity of the core proteins of the hydrophobic cell-associated forms

Heparan sulfate proteoglycans (HSPG) were solubilized from human lung fibroblast monolayers with detergent. Presumptive membrane-associated forms displaying hydrophobic properties were purified by gel filtration on Sepharose CL-4B, by ion-exchange chromatography on Mono Q and by incorporation in lipid vesicles. The HSPG preparations were 125I-iodinated and treated with heparitinase before sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five radiolabeled proteins with apparent molecular weights of 125,000, 90,000, 64,000, 48,000, and 35,000 were visualized by autoradiography. A sixth protein, identified in nonreduced 125I-HSPG preparations, appeared as a non-HS chain-bearing Mr 35,000 peptide which was disulfide-linked to an HS chain-bearing peptide of similar size. This multiplicity of core proteins did not seem to result from proteolysis during the heparitinase treatment itself, since some of the core proteins migrated independently during gel filtration before heparitinase digestion. Moreover, heparitinase digestion of 125I-HSPG purified by affinity chromatography on an immobilized monoclonal antibody yielded only the Mr 64,000 protein. Alternative depolymerizations of the HS chains by heparinase or HNO2 also yielded multiple protein bands. These results imply that heterogeneity of the core protein moiety may be a genuine property of the hydrophobic HSPG of human lung fibroblasts. The occurrence of multiple integral membrane HSPG forms may be relevant for the multiple functions that have been ascribed to cell-surface HSPG.     

An estimation of drag in front crawl swimming

Propulsive arm forces of twelve elite male swimmers during a front crawl swimming-like activity were measured. The swimmers pushed off against grips which are attached to a 23 m tube at 0.8 m under the water surface. The tube was fixed to a force transducer. Since at constant speed, mean propulsive force equals mean drag force this method also provides the mean active drag on a moving swimmer. The mean propulsive force at a speed of v = 1.48 m s-1 appeared to be 53.2 +/- 5.8 N which is two to three times smaller than what is reported by other authors for active drag but which is in agreement with values reported for passive drag on a (towed) swimmer who is not moving. Discrepancies with indirect active drag measurements are discussed.     

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution

We have studied the influence of perturbation of cultured human umbilical vein endothelial cells on the distribution of the von Willebrand factor. As shown previously, short-term (less than 1 hr) treatment of endothelial cells with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) or thrombin resulted in the release of cellular stored von Willebrand factor. Long-term treatment with PMA or thrombin evoked a distinct change in the endothelial cell distribution of von Willebrand factor, evident 24 to 48 hrs after exposure. Whereas the contents of the von Willebrand factor storage sites in the cells were gradually restored within 48 hrs, enhanced amounts of von Willebrand factor were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for von Willebrand factor. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increased after exposure to the phorbol ester, as determined by immunofluorescence microscopy. A second treatment with PMA or thrombin, 48 hrs after cells had been stimulated with these agents, resulted again in the instantaneous release of von Willebrand factor. PMA and thrombin caused a decrease in the von Willebrand factor contents of the extracellular matrix. Pulse-chase experiments revealed that PMA blocked the deposition of von Willebrand factor in the subendothelium, whereas PMA did not affect the degradation of matrix von Willebrand factor. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.     

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.